Alanine Uptake Research Articles

In the absence of counterregulatory hormones, the increase in hepatic glucose production during insulin-induced hypoglycemia in the dog is initiated in the liver rather than the brain.

We have previously demonstrated that the liver can release glucose in response to insulin-induced hypoglycemia, despite the absence of glucagon, epinephrine, cortisol, and growth hormone. The aim of this study was to determine whether this is activated by liver or brain hypoglycemia. We assessed the response to insulin-induced hypoglycemia in the absence of counterregulatory hormones in overnight-fasted conscious adrenalectomized dogs that were given somatostatin and intraportal insulin (30 pmol x kg(-1) x min(-1)) for 360 min. Glucose was infused to maintain euglycemia for 3 h and then to allow limited peripheral hypoglycemia for the next 3 h. During peripheral hypoglycemia, five dogs received glucose via both carotid and vertebral arteries to maintain cerebral euglycemia (H-EU group) concurrently with peripheral hypoglycemia, while six dogs received saline in these vessels to allow simultaneous cerebral and peripheral hypoglycemia (H-HY group). Throughout the study, arterial insulin was 1,675 +/- 295 and 1,440 +/- 310 pmol/l in the H-HY and H-EU groups, respectively. Glucose fell from 6.2 +/- 0.3 to 2.1 +/- 0.0 mmol/l and from 5.8 +/- 0.3 to 1.9 +/- 0.1 mmol/l in the last hour in the H-HY and H-EU groups, respectively (P < 0.05 for both). Norepinephrine rose from 1.12 +/- 0.35 to 2.44 +/- 0.69 nmol/l and from 1.09 +/- 0.07 to 1.74 +/- 0.16 nmol/l in the last hour in the H-HY and H-EU groups, respectively (P < 0.05 for both; no difference between groups). Glucagon, epinephrine, and cortisol were below the limits of detection. The liver switched from uptake to output of glucose during peripheral hypoglycemia in both the H-HY (-7.1 +/- 2.1 to 5.4 +/- 3.1 micromol x kg(-1) x min(-1)) and H-EU (-7.9 +/- 3.5 to 3.4 +/- 1.7 micromol x kg(-1) x min(-1)) groups (P < 0.05 for both; no difference between groups). Alanine levels and net hepatic alanine uptake fell similarly in both groups. There were increases (P < 0.05) in glycerol (12 +/- 3 to 258 +/- 47 micromol/l) and nonesterified fatty acid (194 +/- 10 to 540 +/- 80 micromol/l) levels and in total ketone production (0.4 +/- 0.1 to 1.1 +/- 0.2 micromol x kg(-1) x min(-1)) in the H-HY group, but these parameters did not change in the H-EU group. These data clearly indicate that the lipolytic and hepatic responses to hypoglycemia are driven by differential sensing mechanisms. Thus, during insulin-induced hypoglycemia, when counterregulatory hormones are absent, liver hypoglycemia triggers the increase in hepatic glucose production, whereas cerebral hypoglycemia causes the increases in lipolysis and ketogenesis.

Regulation of the ASC system and Na+/K + pump activities in brown trout (Salmo trutta) hepatocytes

The present study investigates the regulation of Na+/K+ pump activity and alanine uptake in trout hepatocytes. Pump activity increased when cells were incubated in an amino-acid-free medium, while it was reduced in cells from fasted animals. Short-term exposure (3 h) to glucagon modified the activity of the pump in a complex seasonally dependent pattern: in experiments carried out in autumn and winter there was some inhibition, while in spring the pump was activated by this hormone. Pharmacological modification of levels of two intracellular signal transducers, namely cyclic AMP and Ca2+, always led to a reduction in pump activity. These experiments were conducted in May, when activation of the pump by glucagon exposure occurred. There is no apparent explanation for the mechanism by which this hormone modifies the activity of the pump. Glucagon also regulates the activity of system ASC (a Na+-dependent amino acid carrier with short-chain neutral amino acids as preferred substrates). This regulation also showed a seasonally dependent pattern, although the pattern was opposite to that found for the regulation of Na+/K+ pump activity.

Blood-endothelial cell and blood-brain transport of L-proline, alpha-aminoisobutyric acid, and L-alanine.

We report the application of multiple time regression analysis with the in situ brain perfusion technique to measure the rates of passage between blood and brain for [14C] L-proline, [14C] L-alanine, and [14C] alpha-aminoisobutyric acid (AIB) and their rapidly reversible volumes following perfusion of these amino acids from 10 to 60 seconds. We also report on their mechanism of transport. Proline diffused through the blood-brain barrier with a transfer coefficient (Kin) of 0.55 +/- 0.15 x 10(-4) ml/s/g and had no reversible compartment. AIB had a low Kin of 0.68 +/- 0.14 x 10(-4) ml/s/g and a significant reversible volume of 4.34 +/- 0.51 x 10(-3) ml/g in parietal cortex. L-alanine had the highest transfer coefficient, 3.11 +/- 0.26 x 10(-4) ml/s/g, and a reversible volume of 10.03 +/- 0.93 x 10(-3) ml/g in the same cerebral region. Postwash procedures which remove any radiotracer in the vasculature and capillary depletion were performed for alanine and AIB, as they had significant reversible compartments, to test the possibility of rapid efflux from the endothelial cells. Results obtained from wash and capillary depletion procedures suggest that a rapid efflux could occur from endothelial cells after entry of alanine and AIB. Mechanisms of transport for L-alanine and AIB were investigated using amino acids (5 mM) as substrates and inhibitors of different amino acid transport systems. AIB transport was reduced by plasma and L-leucine and unchanged by sodium-free buffer, confirming its passage by the L1 system. L-alanine uptake was sodium-independent and not reduced by plasma. L-serine, L-cysteine, L-leucine and L-phenylalanine produced similar inhibition (66%) while L-alanine produced a lower inhibition (41%). L-arginine increased alanine uptake in cortex and thalamus. Adding L-serine to L-phenylalanine reduced the uptake only in cortex and hippocampus. These data suggest that L-alanine is transported by another L transport system different from the L1 system at the luminal membrane.

Growth hormone pre-treatment combined with exogenous glutamine induced a postoperative shift from glucose to glutamine consumption in the gastrointestinal tract

The aim of this study was to assess whether the protein sparing effects associated with administration of growth hormone (GH) and glutamine in the early post traumatic period deprive the gastrointestinal tract of substrates. Sixteen piglets were randomized to receive GH treatment ( n = 8) for 3 days prior to surgery whilst a control group ( n = 8) received no growth hormone. Organ fluxes of glucose, lactate, pyruvate, alanine and glutamine were measured at 1 and 5 h after surgery. An infusion of glutamine (36 μg/kg/min) was started after the first measurement in both groups. In the GH group (5 h after surgery), hindleg release of glutamine and alanine was found to be lower than in the control group, whilst intestinal glutamine uptake was higher and that of alanine was lower. Hepatic alanine uptake was reduced whilst hepatic glutamine exchange switched from uptake to release. Intestinal glucose consumption was lower in the GH group ( P < 0.05). It is concluded that GH pre-treatment in combination with exogenous glutamine administration induced a shift in gastrointestinal fuel selection which was associated with reduced glucose consumption and increased glutamine consumption. The effect of GH in inducing hepatic release of glutamine compensated for its effect on muscle which results in reduced peripheral glutamine release.

Sodium and Chloride Ion‐Dependent Transport of β‐Alanine Across the Blood‐Brain Barrier

The characteristics of beta-alanine transport at the blood-brain barrier were studied by using primary cultured bovine brain capillary endothelial cells. Kinetic analysis of the beta-[3H]alanine transport indicated that the transporter for beta-alanine functions with Kt of 25.3 +/- 2.5 microM and Jmax of 6.90 +/- 0.48 nmol/30 min/mg of protein in the brain capillary endothelial cells. Beta-[3H]Alanine uptake is mediated by an active transporter, because metabolic inhibitors (2,4-dinitrophenol and NaN3) and low temperature reduced the uptake significantly. Furthermore, the uptake of beta-[3H]alanine required Na+ and Cl- in the external medium. Stoichiometric analysis of the transport demonstrated that two sodium ions and one chloride ion are associated with one beta-alanine molecule. The Na+ and Cl--dependent uptake of beta-[3H]alanine was stimulated by a valinomycin-induced inside-negative K+-diffusion potential. beta-Amino acids (beta-alanine, taurine, and hypotaurine) inhibited strongly the uptake of beta-[3H]-alanine, whereas alpha- and gamma-amino acids had little or no inhibitory effect. In ATP-depleted cells, the uptake of beta-[3H]alanine was stimulated by preloading of beta-alanine or taurine but not L-leucine. These results show that beta-alanine is taken up by brain capillary endothelial cells, via the secondary active transport mechanism that is common to beta-amino acids.

Kinetics, energetics and specificity of a general amino acid transporter from the ectomycorrhizal fungus Paxillus involutus

The kinetics, energetics and specificity of a general amino acid transporter were studied in the ectomycorrhizal fungus Paxillus involutus (Batsch) Fr. The uptake of amino acids showed features characteristic of active transport. After correction for a non-mediated transport component, the kinetics of glutamate, glutamine, alanine and aspartate uptake measured over a wide concentration range followed the simple Michaelis-Menten saturation curves. The apparent K m derived from the Eadie-Hofstee plots ranged from 7 μM for alanine to 27 μM for glutamate. Dinitrophenol, carbonyl cyanide m-chlorophenylhydrazone and NaN3 strongly inhibited amino acid uptake, whereas dicyclohexylcarbodiimide, vanadate and the ionophores monensin and nonactin had no effect on the uptake. Both pH dependence and inhibition by protonophores are consistent with a proton symport mechanism for amino acid uptake by P. involutus. Competition studies indicated a broad substrate recognition by the uptake system, which resembles the general amino acid permease of yeast. Dixon plots of the inhibition of glutamate uptake by alanine, lysine and methionine sulfoximine showed that inhibitions were competitive. The physiological importance of this transporter for the exchange of nitrogenous compounds between fungal and host plant cells in ectomycorrhizal associations is discussed.

Gluconeogenesis in the livers of diet-restricted rats--a 13C nuclear magnetic resonance study.

Gluconeogenic activity is reduced during starvation. However, it is less clear whether the utilization of gluconeogenic substrates is diminished with mild but prolonged diet restriction and, if so, whether there are intrinsic changes in the gluconeogenic pathway. We examined gluconeogenesis in the livers of diet-restricted rats with 13C nuclear magnetic resonance (NMR) spectroscopy. Fischer 344 rats were given 88% (DR group) of what was consumed by the weight-matched ad libitum-fed normal rats (CL group). At the end of 5 weeks, the removed livers were perfused with [3-13C] alanine while 13C NMR spectroscopy was performed. The final body and liver weights were the same for the two groups. In DR rats, both intrahepatic [3-13C] alanine and metabolites generated via pyruvate and oxaloacetate, including aspartate and carbamoyl aspartate, appeared in significantly reduced amounts. There was also marked diminution in the production of glucose. In the livers of DR rats, alanine uptake through System A transport, the fluxes through pyruvate carboxylase, the biosynthesis of pyrimidine nucleotides, and the production of glucose from alanine were all significantly decreased with mild intake restriction. Attenuated protein synthesis in the liver of diet-restricted animals may be the cause for this decreased utilization of alanine for gluconeogenesis.

Osmotic regulation of intracellular solute pools in Lactobacillus plantarum.

Bacteria respond to changes in medium osmolarity by varying the concentrations of specific solutes in order to maintain constant turgor pressure. The cytoplasmic pools of K+, proline, glutamate, alanine, and glycine of Lactobacillus plantarum ATCC 14917 increased when the osmolarity of the growth media was raised from 0.20 to 1.51 osmol/kg by KCL. When glycine-betaine was present in a high-osmolarity chemically defined medium, it was accumulated to a high cytoplasmic concentration, while the concentrations of most other osmotically important solutes decreased. These observations, together with the effects of glycine-betaine on the specific growth rate under high-osmolarity conditions, suggest that glycine-betaine is preferentially accumulated in L. plantarum. Uptake of glycine-betaine, proline, glutamate, and alanine was studied in cells that were alternately exposed to hyper- and hypo-osmotic stresses. The rate of uptake of proline and glycine-betaine increased instantaneously upon increasing the osmolarity, whereas that of other amino acids did not. This activation occurred also under conditions in which protein synthesis was inhibited was most pronounced when cells were pregrown at high osmolarity. The duration of net transport was a function of the osmotic strength of the assay medium. Glutamate uptake was not activated by an osmotic upshock, and the uptake of alanine was low under all conditions tested. When cells were subjected to osmotic downshock, a rapid efflux of accumulated glycine-betaine, proline, and alanine occurred whereas the pools of other amin acids remained unaffected. The results indicate that osmolyte efflux is, at least to some extent, mediated via specific osmotically regulated efflux systems and not via nonspecific mechanisms as has been suggested previously.

Kinetics and Specificity of a H+/Amino Acid Transporter from Arabidopsis thaliana

The amino acid transporter AAP1/NAT2 recently cloned from Arabidopsis thaliana was expressed in Xenopus oocytes, and we used electrophysiological, radiotracer flux, and electron microscopic methods to characterize the biophysical properties, kinetics, and specificity of the transporter. Uptake of alanine was H(+)-dependent increasing from 14 pmol/oocyte/h at 0.032 microM H+ to 370 pmol/oocyte/h at 10 microM H+. AAP1 was electrogenic; there was an amino acid-induced depolarization of the oocyte plasma membrane and net inward currents through the transporter due to the transport of amino acids favoring neutral amino acids with shortside chains. The maximal current (imax) for alanine, proline, glutamine, histidine, and glutamate was voltage and [H+]o-dependent. Similarly, the imaxH was voltage and [amino acid]o-dependent. The imax for both H+ and amino acid were dependent on the concentrations of their respective cosubstrates, suggesting that both ligands bind randomly to the transporter. The K0.5 of the transporter for amino acids decreased as [H+]o increased and was lower at negative membrane potentials. The K0.5 for H+ was relatively voltage-independent and decreased as [amino acid]o increased. This positive cooperativity suggests that the transporter operates via a simultaneous mechanism. The Hill coefficients n for amino acids and H+ were > 1, suggesting that the transporter has more than one binding site for both H+ and amino acid. Freeze-fracture electron microscopy was used to estimate the number of transporters expressed in the plasma membrane of oocytes. The density of particles on the protoplasmic face of the plasma membrane of oocytes expressing AAP1 increased approximately 5-fold above water-injected controls and corresponded to a turnover number 350 to 800 s-1.

Effect of epidermal growth factor on placental amino acid transport and regulation of epidermal growth factor receptor expression of hepatocyte in rat.

We investigated the effect of epidermal growth factor (EGF) on amino acid transport in the rat placenta and the hormonal and nutritional regulation of hepatic expression of epidermal growth factor receptor (EGFR). Fetuses born to maternal rats administered EGF were larger than control fetuses, while fetuses born to mothers administered EGF antibody were smaller than controls. The fetomaternal amino acid concentration ratio (fetal blood/maternal blood) was higher in the EGF-treated group than in the control group, and was lower in the EGF antibody-treated group. EGF treatment stimulated 14C-aminoisobutyric acid, isoleucine and alanine uptake by placental explants as did treatment with insulin-like growth factor-1 and insulin, which have been reported to stimulate the active amino acid transport. Thus, EGF promoted amino acid transport in the rat placenta and influenced fetal growth as a result. In addition, retinol induced a 170% increase of EGF binding to rat hepatocytes and insulin induced a 120% increase of EGF binding, while amino acids (isoleucine and serine) had no effect on EGF binding. This result indicated that there is hormonal regulation of EGF binding to hepatocytes and that changes of some hormone levels may affect hepatic EGFR expression.

Adaptive regulation of amino acid transport in nutrient-deprived human hepatomas

Malignant cells require increased amounts of amino acids, in particular glutamine and leucine, to support DNA and protein biosynthesis. Although plasma concentrations in the center of solid tumors can be much lower than normal circulating levels, it is still unknown how tumor cells can survive despite low amino acid levels. We examined the effects of glutamine or leucine deprivation on cell growth and amino acid transport activity in two human hepatoma cell lines, SK-Hep and HepG2. We studied the transport of glutamine, leucine, alanine, and arginine. The carrier-mediated uptake of 3H-amino acids was determined in cells cultured in normal and amino acid-deprived media. The growth of both cell lines was dependent on the concentration of glutamine and leucine. In SK-Hep, there was a significant increase in initial rate glutamine transport activity in the glutamine-deprived group, attributable to an increase in transporter affinity (Km; 0.6 mmol/L [control], 385 +/- 43 mumol/L; 0.1 mmol/L, 221 +/- 11 mumol/L; P < 0.01). At low glutamine concentration, the saturable Na(+)-independent uptake of leucine and arginine as well as the Na(+)-dependent uptake of alanine increased significantly in both SK-Hep and HepG2. Similarly, in leucine-deprived SK-Hep cells, leucine uptake increased twofold, but the change was attributable to an enhanced transporter capacity (Vmax; 0.2 mmol/L [control], 38,900 +/- 700; 0.0 mmol/L, 75,900 +/- 4,900 pmol/mg protein per minute; P < 0.001). Adaptive increases in initial rate amino acid transport activities were elicited by glutamine and leucine deprivation in these two human hepatoma cell lines. Decreased extracellular amino acid levels encountered by tumors in vivo may elicit similar adaptive responses that contribute to the maintenance of cytoplasmic levels of amino acids essential for growth.

Enhanced hepatic gluconeogenic capacity for selected precursors after endurance training.

The effects of endurance training (running 90 min/day, 30 m/min, approximately 10% grade) on hepatic gluconeogenesis were studied in 24-h-fasted rats by using the isolated liver perfusion technique. After isolation, livers were perfused (single pass) for 30 min with Krebs-Henseleit bicarbonate buffer and fresh bovine red blood cells (hematocrit 20-24%) with no added substrate. Alanine (10 mM), dihydroxyacetone (20 mM), or glutamine (10 mM) was then added to the reservoir, and perfusions continued for 60 min. No significant differences were observed in perfusate pH, hematocrit, bile production, or serum alanine aminotransferase effluxing from livers from trained or control animals for any perfusion. Livers from trained animals that were perfused with 10 mM alanine demonstrated significantly higher rates of glucose production compared with livers from control animals (0.51 +/- 0.04 vs. 0.40 +/- 0.02 micromol.min-1.g liver-1, respectively). Elevations of a similar magnitude were observed for rates of [14C]alanine incorporation into [14C]glucose in livers from trained vs. control animals (8,797 +/- 728 vs. 6,962 +/- 649 dpm.min-1.g liver-1, respectively). Significant increases were also observed in hepatic alanine uptake (30%), oxygen consumption (23%), urea release (22%), and 14CO2 production (29%) of livers of endurance-trained animals. In contrast, no significant differences between groups were observed for hepatic glucose output after perfusions with either dihydroxyacetone (1.75 +/- 0.06 micromol.min-1.g liver-1) or glutamine (0.62 +/- 0.04 micromol.min-1.g liver-1). Further, during perfusions with dihydroxyacetone and glutamine, training had no significant impact on precursor uptake, oxygen consumption, or urea output. The current findings indicate a training-induced adaptation for hepatic gluconeogenesis located below the level of the triose phosphates.

Microcystin Uptake and Inhibition of Protein Phosphatases: Effects of Chemoprotectants and Self-Inhibition in Relation to Known Hepatic Transporters

The microcystins (Mcyst) are cyclic peptide hepatotoxins produced by cyanobacteria. They are chemically very stable and represent a public health threat when they occur in water bodies used for human consumption. Mice injected ip with Mcyst (LD50 50-100 μg/kg) accumulate Mcyst in the liver and die within 2-4 hr with massive intrahepatic hemorrhage. Pretreatment of mice with cyclosporin A (CP), rifamycin (Rif), trypan blue (TB), and trypan red (TR) protected the animals from a lethal dose of the toxin. The studies reported here using the freshly isolated rat hepatocyte model were undertaken in order (1) to evaluate the contribution of Mcyst transport in hepatocytes to the mechanism of chemoprotection for Mcyst in vivo toxicity by CP, Rif, TB, and TR and (2) to better characterize the hepatic Mcyst transporter in this model and determine its relationship to other bile acid/organic anion transporters that have already been fully described. Incubations with 125I-Mcyst were used to measure Mcyst uptake and accumulation in hepatocytes. It has been shown that at the cellular level Mcyst binds to and inhibits protein phosphatases 1 and 2A (PP) at nanomolar concentrations. Lethal doses of Mcyst in mice resulted in rapid profound inhibition of hepatic PP activity. PP activity was also inhibited in hepatocytes incubated with 100-500 nM Mcyst. PP inhibition in these studies was used as a marker of metabolic effects of the toxin. The chemoprotectants CP (5 μM), Rif(50 μM), TR (20 μM), and TB (20 μM) decreased accumulation of Mcyst (320 nM, a toxic concentration) after 30 min incubation to 37, 26, 30, and 66%, respectively, of that of cells treated with Mcyst only. Inhibition of PP activity in these cells was decreased. Inhibition of PP activity in hepatocytes was also decreased by known inhibitors of Mcyst transport: 50 μM of the bile acids cholate and taurocholate (TC) and 50 μM sulfobromophthalein. For all compounds tested the amount of Mcyst accumulated in the hepatocytes correlated qualitatively with the extent of PP inhibition. From these results it can be concluded that inhibition of Mcyst uptake by hepatocytes is the most likely mechanism of chemoprotection for Mcyst in vivo toxicity for TR, TB, CP, and Rif. Uptake of Mcyst was unaffected by changes in the ionic composition of the uptake buffer but was significantly decreased when PP activity of hepatocytes was inhibited by preincubation with Mcyst. This effect resulted directly from PP inhibition since preincubation with calyculin A (a cell permeant PP inhibitor, chemically unrelated to Mcyst) also led to inhibition of Mcyst uptake. In contrast, alanine and TC uptake were unaffected in cells with PP activity fully inhibited, indicating that the activities of these latter transporters, unlike that for Mcyst, are probably not controlled by serine/threonine phosphorylation. The pattern of inhibition, the lack of sodium or chloride dependency, and the sensitivity to PP inhibition indicate that the hepatic transport of Mcyst is unlikely to be via one of the already fully characterized sinusoidal bile salt organic anion transporters.

In vivo amino acid fluxes in regenerating liver after two-thirds hepatectomy in the rat

Background/Aims: Recent reports in the literaturesuggest that liver cell swelling following amino acid influx exerts anabolic and anti-catabolic effects. We have tested the possibility that rapid liver growth after partial hepatectomy is promoted by an increased amino acid-influx and that this is associated with an increased hepatic water content and a decreased rate of proteolysis. Methods: Two-thirds hepatectomy was performed in rats. Plasma liver flow and amino acid fluxes were measured after 24 or 48 h. Results: Plasma liver flow was increased 24 and 48 h after partial hepatectomy or sham-operation in pair-fed animals. At these time points, in both groups there was a specific two- to threefold increased net hepatic uptake of the amino acids alanine and glycine, both being transported by the sodium-coupled amino acid transport system A/ASC. No changes in uptake of system N transported amino acids were observed. Both in partially hepatectomized and sham-operated pair-fed animals, the hepatic uptake of alanine and glycine was accompanied by a minor increase in tissue water (from 68 to 70%). Proteolysis, measured by leucine efflux, was only reduced in regenerating livers. Conclusions: We conclude that cell swelling is not animportant factor in the stimulation of net protein synthesis during liver regeneration.

In vivo amino acid fluxes in regenerating liver after two-thirds hepatectomy in the rat

<h2>Abstract</h2><h3>Background/Aims:</h3> Recent reports in the literaturesuggest that liver cell swelling following amino acid influx exerts anabolic and anti-catabolic effects. We have tested the possibility that rapid liver growth after partial hepatectomy is promoted by an increased amino acid-influx and that this is associated with an increased hepatic water content and a decreased rate of proteolysis. <h3>Methods:</h3> Two-thirds hepatectomy was performed in rats. Plasma liver flow and amino acid fluxes were measured after 24 or 48 h. <h3>Results:</h3> Plasma liver flow was increased 24 and 48 h after partial hepatectomy or sham-operation in pair-fed animals. At these time points, in both groups there was a specific two- to threefold increased net hepatic uptake of the amino acids alanine and glycine, both being transported by the sodium-coupled amino acid transport system A/ASC. No changes in uptake of system N transported amino acids were observed. Both in partially hepatectomized and sham-operated pair-fed animals, the hepatic uptake of alanine and glycine was accompanied by a minor increase in tissue water (from 68 to 70%). Proteolysis, measured by leucine efflux, was only reduced in regenerating livers. <h3>Conclusions:</h3> We conclude that cell swelling is not animportant factor in the stimulation of net protein synthesis during liver regeneration.

Factors affecting amino acid uptake by the ectomycorrhizal fungus Paxillus involutus

Factors affecting amino acid uptake by the ectomycorrhizal fungus Paxillus involutus were investigated using tracer kinetic experiments. Amino acid uptake by P. involutus was dependent on the pH of the incubation medium. Uptake of L-glutamate and L-glutamine had a distinct pH optimum around pH 4.0 and declined sharply between pH 4.5 and 6.4. Uptake of alanine and aspartate was also highest at low pH values (3-4) but declined more slowly with increasing pH. Total uptake of labelled amino acids was unaffected by 0.05 or 0.5 mM ammonium while 5 mM ammonium decreased amino acid uptake. Nitrate or glucose did not significantly affect amino acid uptake but glucose greatly increased the proportion of carbon that was respired. Uptake of the different amino acids decreased by a factor of 4 to 10 with increasing age (1-5 wk) and the proportion of absorbed C-14 that was respired increased with age. Changes in the size of the total amino acid pool were inversely related to those in the rate of uptake and the pool increased steadily with age. The implications of the ability of P. involutus to take up amino acids are discussed. (Less)

Intestinal nutrient transport in genetically obese mice

The small intestine of obese animals supplies nutrients to a metabolic live mass (body weight 0.75) that is much higher than that of age- and sex-matched lean animals. To determine the mechanisms of adaptation of the small intestine to obesity, we determined the rate of uptake of D-glucose and five amino acids, the site density of intestinal D-glucose transporters, and the permeability of the absorptive mucosa in isolated everted intestinal sleeves of genetically obese male mice (C57BL/6J ob/ob) and their lean male littermates. Intestinal D-glucose, proline, alanine, aspartate, leucine, and lysine uptakes per milligram were each similar in the small intestine of obese and control mice. Mucosal permeability, site density of intestinal D-glucose transporters, and their affinity for phlorizin were also each similar between obese mice and their lean controls. In contrast, intestinal D-glucose, proline, alanine, aspartate, leucine, and lysine uptakes per centimeter of small intestine were each approximately 40% greater in obese mice compared with lean controls (P < 0.001 to P < 0.08, depending on the nutrient and intestinal region). Differences in total absorptive capacity for any nutrient between the small intestine of obese and lean mice reflect mainly differences in intestinal weights. Thus, genetic obesity is associated with increased intestinal growth, which augments absorption of all types of nutrients. The ratio of intestinal absorptive capacity to metabolic mass, and of intestinal mass to metabolic mass, does not change with obesity, indicating that in mice, changes in intestinal mass and in absorptive capacity are directly proportional to changes in metabolic mass.

The Time Course of Adaptation of Intestinal Nutrient Uptake in Mice Is Independent of Age

Slow adaptive responses are typical of aged animals. The pace of intestinal adaptation is relatively rapid in young mice but is not known in aged mice. In this study, we determined the time course of adaptation of intestinal nutrient uptake to abrupt and substantial changes in levels of dietary nutrients in young and aged mice. Mice (24-mo-old and 6- to 7-mo-old, controls) were fed a no carbohydrate, high protein diet for 7 d and then were switched to a high carbohydrate, low protein diet for 0, 1, 2, 3 and 5 d. Body weights, feeding rates, intestinal weights and lengths were all independent of diet. Within 1 d after the switch in diet, glucose and fructose uptakes increased in young and aged mice. In both age groups, alanine and aspartate uptakes decreased significantly, whereas proline, leucine and lysine uptakes were independent of diet. In both age groups, total intestinal absorptive capacity for glucose and fructose increased overnight by about 70 and 200%, respectively. Previous studies linked time course of adaptation to rates of enterocyte proliferation. Rates of enterocyte turnover and migration along the crypt/villus axis of proximal, middle and distal small intestine were independent of age. Thus, the time course of intestinal adaptation in aged mice remains largely unchanged from that in young mice, probably because rates of enterocyte proliferation seem independent of age.

Ontogeny of L-alanine uptake in plasma membrane vesicles from rat liver.

Alanine uptake into liver plasma membrane vesicles was studied at different stages of postnatal rat development. Before weaning, alanine hepatic uptake showed lower values for the global KM than after weaning (0.34, 0.77, 1.45, and 1.61 mM for 1-, 15-, and 28-d-old and adult rats, respectively). Alanine uptake capacity increased progressively until reaching maximum values in the adult state (values for Vmax: 0.078, 0.199, 0.317, and 0.613 nmol alanine/mg protein/3 s for 1-, 15-, and 28-d-old and adult rats, respectively). These results seem to point to a prevalence of a high affinity, low capacity alanine transport component (traditionally assumed to be attributable to system A) in newborn and suckling rats, in agreement with our previous results on isolated hepatocytes (Martínez-Mas JV, Casado J, Felipe A, Marin JJG, Pastor-Anglada M: Biochem J 293: 819-824, 1993). The suckling-weaning developmental transition seems to play a role in establishing the pattern of adult hepatic alanine transport characterized by a higher capacity but a lower affinity (because most alanine is taken up by system ASC) inasmuch as KM values show a 100% increase after weaning, although Vmax values continue to increase steadily until the adult age.

Plasma disposal rate and hepatic alanine metabolism in pregnant and nonpregnant rabbits.

To better define the glucose-alanine relationship and hepatic alanine metabolism during pregnancy, glucose and alanine turnover rates (TR) and simultaneous hepatic alanine uptake were determined in nine term pregnant (P) and 12 nonpregnant (NP) rabbits who had undergone chronic catheterization of both the systemic and hepatic circulations. [U-14C3]Alanine and [3-3H1]glucose were used as tracers. Whole animal [U-14C3]alanine TR was significantly greater in P than in NP animals (42.7 +/- 1.7 versus 31.9 +/- 1.8 mumol.min-1, respectively, p < 0.001). However, there was no difference when the alanine TR was compared on a weight-specific basis (11.3 +/- 0.6 versus 9.6 +/- 0.6 mumol.min-1.kg-1). The glucose TR was higher in P compared with NP rabbits on both an absolute (122.3 +/- 6.2 versus 79.8 +/- 5.8 mumol.min-1, p < 0.001) and a weight-specific basis (32.9 +/- 1.4 versus 24.0 +/- 1.9 mumol.min-1.kg-1, p < 0.005). Although the fraction of plasma glucose derived from plasma alanine in the whole animal was similar in P and NP animals (16.7% +/- 1.4 versus 13.9 +/- 0.7%, respectively), the absolute amount of plasma glucose derived from plasma alanine was greater in P rabbits (20.8 +/- 1.8 versus 11.2 +/- 1.2 mumol.min-1, p < 0.0005). In addition, the percentage of plasma alanine being converted to plasma glucose was greater in P than in NP animals (48.1 +/- 2.6 versus 34.6 +/- 2.0%, p < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)